Wild type activity of the adenovirus 5 E1b gene promoter requires transcriptional readthrough from the upstream E1a gene. This cis-dominant property of early genomes is relieved with the onset of viral DNA replication. The goal of this proposal is to determine whether transcription termination helps define a transcriptionally active domain and to analyze the mechanism. The central hypothesis is that readthrough transcription activates a downstream promoter by facilitating access to a distal enhancer sequence. The first specific aim is to employ DNA footprinting techniques in isolated nuclei to determine whether termination quantitatively or qualitatively alters pre- initiation complex formation at the E1B promoter.
For specific aims 2 and 3, recombinant adenoviruses will be constructed, and gene expression quantified, to examine further the consequences for viral gene expression of artificial termination.
Aim 2 will determine whether termination interferes with activity of a distal enhancer required for E1b promoter function. E1a element II, a global enhancer of early viral gene transcription, will be inactivated in viral strains with functional or inactivated termination sequences. The suppression of termination-dependent inhibition of downstream gene expression will be tested by substituting a strong downstream enhancer-promoter for E1b.
Aim 3 will determine whether expression of other early genes can be inhibited by one or more genomic insertions of termination sequences. Finally, specific aim 4 will determine whether termination of transcription affects the activity of downstream promoters in a cellular genome. A simple drug selection and reporter screen will allow simultaneous assay of many independently derived stable clones transfected with expression cassettes that mimic the functional arrangements of sequences in the recombinant viruses. The investigation of the relationship between transcription termination and transcriptionally active domains in an authentic viral system provides a unique opportunity to acquire new knowledge about an important but still poorly understood aspect of gene expression. Successful accomplishment of the aims also will provide evidence for a previously unrecognized activity governing domains of cellular gene expression. Understanding new molecular mechanisms for controlling gene expression has applications in the development of viral vectors and cellular expression systems for gene therapy.
| Shen, Li; Spector, David J (2010) Read-through activation of transcription in a cellular genomic context. PLoS One 5:e15704 |
| Shen, Li; Spector, David J (2003) Local character of readthrough activation in adenovirus type 5 early region 1 transcription control. J Virol 77:9266-77 |
| Spector, David J; Johnson, Jeffrey S; Baird, Nicholas L et al. (2003) Adenovirus type 5 DNA-protein complexes from formaldehyde cross-linked cells early after infection. Virology 312:204-12 |
